Processes have already been established for producing interferon, growth hormones and various other polypeptides by gene technology using host cells of Escherichia coli, Bacillus subtilis, yeast and the like. However, these processes, although established, have some problems still remaining to be solved, and it is not always easy to prepare polypeptides which are exactly identical with those naturally occurring. One of the most serious problems of these is that it is difficult to obtain polypeptides with an N terminus which is identical in structure to the corresponding terminus of natural products. When the genes coding for these polypeptides are to be expressed directly in host cells, the translation of the gene into the polypeptide usually takes place first at the start codon ATG, so that the polypeptide expressed has a formylmethionine residue at its N terminus. Natural polypeptides are not very likely to have formylmethionine at the N terminus, and accordingly, this method is not usable for preparing polypeptides having other N terminus. The formylmethionine residue can be removed from the polypeptide by a chemical reaction using cyanogen bromide, but when the polypeptide has other methionine residues at positions other than the N terminus, the polypeptide chain itself is cut at the position when the above method is resorted to. It is therefore impossible to obtain the desired polypeptide.
A method is also known in which the gene coding for the desired polypeptide is linked with the gene coding for some other polypeptide to express a fused polypeptide. With this method, the fusion polypeptide obtained should be treated with enzymes such as trypsin or chemically treated using cyanogen bromide to separate the desired polypeptide from the fused product. Nevertheless, if the desired polypeptide chain includes amino acid sequence which is a target for the enzyme or chemical used, the peptide chain is cut at the position of the amino acid sequence with the result that the method fails to afford the desired polypeptide. Even if the desired polypeptide can be separated from the fused polypeptide by the above method, the isolation of the desired polypeptide usually requires two purification steps, i.e., the step of separating the fused polypeptide from the resulting crude cell extract and the step of separating the desired polypeptide from the reaction mixture containing the separated fused polypeptide. These steps need cumbersome procedures and invariably result in a low yield.
Thus, the conventional genetic engineering methods encounter great difficulties in preparing polypeptides which are exactly identical with natural products. It is desired in the art to carry out research on and develop improved methods by which polypeptides which are exactly identical with those of natural origin can be prepared directly from host cells.
An object of the present invention is to provide an improved process, as desired in the art, for directly producing from host cells a polypeptide which is exactly identical with the natural one. Another object of the present invention is to provide a novel vector and a microorganism having the vector, for practicing this process.